**3.1.2 Results and discussion**

200 Biogas

*Kluyveromyces marxianus* 499 obtained from Institute of Agricultural and Food Biotechnology Warsaw, Poland, in lyophilized form was used in all experiments. The yeast strain was cultivated on plates prepared with Wort Agar growth media from Merck Company Darmstadt (Germany) with the addition of 3% lactose using an incubator shaker under sterile conditions at pH 4.5 and a temperature 25ºC for 48 h. The yeast was aseptically transferred from the plates into 300 ml cultivation flasks containing 100 ml of Wort Agar medium from Merck Company Darmstadt (Germany) supplemented with 3% lactose, and cultivated at 25ºC for 24 h on a rotatory shaker. The yeast culture was immobilized and suspended in 2% w/v sodium alginate and then added drop-wise to 1.5% w/v CaCl2 solution. The CaCl2 was decanted. The beads were used for inoculation of experimental

A solution of dried permeate from UF whey permeate from the Dairy Plant in Wolsztyn, Poland, was used as a substrate in this study. The solution was prepared by dissolving dried permeate in distilled warm water to obtain 50 g L-1 lactose concentration in wastewater,

Fermentation process was carried out in three UASB reactors with an active volume of 5 L. There were the gas-liquid-solid (G-L-S) separators on the top of each reactor. Whey permeate solution was pumped continuously to the bottom part of the reaction tank by means of the peristaltic pump. The necessary mixing was achieved through the upward wastewater flow and a stirrer operated at 40 rpm. The reactors were water-jacketed and operated at a constant temperature of 25ºC ± 1°C. The pH of mixed liquid in the reactors

For start-up of continuous culture, 1 L of the beads culture medium were grown at 25C for 24 h in a 2 L Erlenmeyer flask filled with 0.5 L of UF whey permeate after heat sterilization (120C, 20 min). The concentration of lactose in whey permeate was 50 g L-1. Mixing was achieved by stirring with a magnetic stirrer at 200 rpm. The cell suspension was then aseptically transferred to each UASB reactor which was kept in batch operation for 24 h before switching on the continuous feeding. The reactors were operated at the HRTs of 12, 24 and 48 h. At each HRT the reactors were operated till they had reached the steady-state (the steady-state conditions were evidenced when the standard deviations of the ethanol

Lactose concentrations and ethanol concentrations in the effluent distillate were determined according to Standard Methods (PN-67/A-86430; PN-A-79528-3:2007). The biomass concentration of yeast (dry matter) was calculated according to Standard Methods (P-78/C-04541). The samples were analyzed in triplicate and results were reproducible within 3%

concentrations and lactose concentrations in the effluent distillate were within 3%).

**3.1 Bioethanol production by** *Kluyveromyces marxianus*

**3.1.1.2 Fermentation medium and experimental system** 

was controlled automatically at pH 4.76 – 4.86 with 2 M NaOH.

**3.1.1 Materials and methods** 

while the initial COD was 56 g L-1.

**3.1.1.3 Analytical methods** 

standard deviation.

**3.1.1.1 Microorganisms** 

reactors.

The effects of HRTs on the lactose concentration in the effluent distillate and percent lactose consumption are shown in Fig. 5. When the HRT was 12 h, the average lactose concentration in the effluent distillate was as high as 25 g L-1 and the average lactose utilization efficiency was only 50%. Increasing the HRT from 12 to 24 h increased the average yield of lactose utilization to 85%. Further increase in the HRT from 24 to 48 h resulted in the highest lactose utilization of 95%. Similar results obtained Ghaly & El-Taweel (1997). They observed 98% lactose utilization for continuous fermentation from cheese whey with 50 g L-1 initial lactose concentration at the HRT of 42 h using the yeast strain of *Candida pseudotropicalis*. Kargi & Ozmihci (2006) reported complete fermentation of lactose (35 g L-1 initial lactose concentration) in cheese whey powder (CWP) solution using the yeast strain of *K. marxianus* at HRT of 48 h. Zafar & Owais (2006) obtained about 86% lactose utilization from crude whey within 22 h by *K. marxianus*.

Fig. 5. Effects of HRT on the lactose concentration in the effluent and percent lactose consumption

According to Ghaly & El-Taweel (1997) lower lactose fermentation efficiency under low HRT could be attributed to the cell washout phenomenon and the low cell numbers in the reactor chamber. To remove this problem, during this experiment, the reactors were provided with G-L-S separator and the immobilization of yeast culture was done. The immobilization process made ethanol production more efficient compared to the free system and prolonged the activity of yeast cells (Kourkoutas et al. 2004), which is especially important in continuous fermentation processes. Moreover, the application of immobilization process reduced the risk of microbial cells infection when the yeasts were cultivated on the fermentation medium that was not sterilized before use (Lewandowska & Kujawski, 2007).

Feasibility of Bioenergy Production from

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

and 48 h, respectively.

**3.1.3 Conclusions** 

Fig. 7. Effect of HRT on the ethanol yield.

while the ethanol yield was 0.325 g g-1.

**3.2.1 Materials and methods** 

**3.2.1.1 Microorganisms** 

**3.2 Bioethanol production by** *Saccharomyces cerevisiae* 

Ethanol yield (g g-1)

Ultrafiltration Whey Permeate Using the UASB Reactors 203

12 24 48

HRT (h)

Ozmihci & Kargi (2007) stated, that biomass concentration is an important parameter affecting the ethanol formation efficiency. The volumetric rate of sugar utilization can increase with biomass concentration. They found that when the biomass concentration was 510 mg L-1, the rate of sugar utilization was about 1580 mg L-1 h-1 at HRT of 30 h. The maximum sugar utilization rate of 2200 mg L-1 h-1 they obtained at the biomass concentration of 1020 mg L-1 and HRT of 24 h. In this study the concentration of yeast in the UASB reactors ranged from 705 to 869 mg L-1 by the duration of the experiment. The volumetric rate of sugar utilization was as high as 927, 1576, 1724 mg L-1 h-1 at HRT of 12, 24

The utilization of whey UF permeate to ethanol in continuous fermentation is possible. *Kluyveromyces marxianus* was able to metabolize lactose and the total fermentation efficiency was as high as 95%. The lactose utilization and ethanol production were connected with the HRT. The maximum daily ethanol production of 8.61 g L-1d-1 was achieved when the HRT was 24 h and the ethanol yield was 0.203 g g-1. The results indicated that the HRT should be 24 h to obtain high rates of ethanol formation and to avoid product inhibition. Doubling the HRT to 48 h did not contribute to a noticeable increase of ethanol production and the daily ethanol production decreased to 7.73 g L-1d-1

The yeast *Saccharomyces cerevisiae* B-4 obtained from Institute of Agricultural and Food Biotechnology Warsaw, Poland, was used for assessment ultrasound exposition to ethanol production. The yeast cultures were cultivated on YPG slants (2% glucose, 2% peptone, 1% yeast extract) supplemented with 2% agar, at pH 5.0 and 30 ºC for 24 h. The active cultures

Fig. 6 shows the variations of daily ethanol production and ethanol concentration with the initial lactose concentration of 50 g L-1. The maximum daily ethanol production of 8.61 g L-1 d-1 was obtained at the HRT of 24 h. Increasing in the HRT to 48 h decreased daily ethanol production to the average value of 7.73 g L-1 d-1 in spite of the fact that alcohol concentration increased from 8.61 to 15.45 g L-1. When the HRT was 12 h, the average daily ethanol production was 4.46 g L-1 d-1, while the average ethanol concentration was as low as 2.24 g L-1. The results were similar to the ones obtained by Kourkoutas et al. (2002). The ethanol productivity was 7.0 and 8.0 g L-1 d-1 at the HRT of 25 and 20 h respectively, using whey as a substrate fermentation and immobilized cells of *K. marxianus*.

Fig. 6. Effects of HRT on daily ethanol production and ethanol concentration in the effluent.

The negative effect of longer HRT on the daily ethanol productivity could be associated with a negative effect of increasing concentration of ethanol. According to Golubev & Golubev (2004) ethanol concentration of 2 - 4% produces a negative effect on the growth of *Kluyveromyces*. Silveira et al. (2005) observed the growth inhibition of *K. marxianus* when the ethanol concentration increased from 10 g L-1 to 20 g L-1. de Gultz (2009) studied alcohol tolerance of direct whey fermenting yeasts (four strains of *K. marxianus*) and indirect whey fermenting yeasts (three strains of *S. cerevisiae*). From the results it can be seen that some strains of *K. marxianus* showed considerable alcohol tolerance of 71 - 81 g L-1 with fermentation times ranging from 11 to 32 h , while alcohol tolerance for *S. cerevisiae* reached 85 - 148 g L-1 with fermentation time ranging from 29 to 64 h.

Increasing in the HRT, increased the ethanol yield (g ethanol g-1 consumed lactose), (Fig. 7). The ethanol yield obtained in this study was 0.089, 0.203, 0.325 g g-1 at the HRT of 12, 24, 48 h, respectively. Silveira et al. (2005) obtained a higher ethanol yield of 0.52 g g-1 with the initial lactose concentration of 50 g L-1 with the yeast strain of *K. marxianus*.

Fig. 6 shows the variations of daily ethanol production and ethanol concentration with the initial lactose concentration of 50 g L-1. The maximum daily ethanol production of 8.61 g L-1 d-1 was obtained at the HRT of 24 h. Increasing in the HRT to 48 h decreased daily ethanol production to the average value of 7.73 g L-1 d-1 in spite of the fact that alcohol concentration increased from 8.61 to 15.45 g L-1. When the HRT was 12 h, the average daily ethanol production was 4.46 g L-1 d-1, while the average ethanol concentration was as low as 2.24 g L-1. The results were similar to the ones obtained by Kourkoutas et al. (2002). The ethanol productivity was 7.0 and 8.0 g L-1 d-1 at the HRT of 25 and 20 h respectively, using whey as a

12 24 48

Ethanol concentration Daily ethanol production

HRT (h)

Fig. 6. Effects of HRT on daily ethanol production and ethanol concentration in the effluent.

The negative effect of longer HRT on the daily ethanol productivity could be associated with a negative effect of increasing concentration of ethanol. According to Golubev & Golubev (2004) ethanol concentration of 2 - 4% produces a negative effect on the growth of *Kluyveromyces*. Silveira et al. (2005) observed the growth inhibition of *K. marxianus* when the ethanol concentration increased from 10 g L-1 to 20 g L-1. de Gultz (2009) studied alcohol tolerance of direct whey fermenting yeasts (four strains of *K. marxianus*) and indirect whey fermenting yeasts (three strains of *S. cerevisiae*). From the results it can be seen that some strains of *K. marxianus* showed considerable alcohol tolerance of 71 - 81 g L-1 with fermentation times ranging from 11 to 32 h , while alcohol tolerance for *S. cerevisiae* reached

Increasing in the HRT, increased the ethanol yield (g ethanol g-1 consumed lactose), (Fig. 7). The ethanol yield obtained in this study was 0.089, 0.203, 0.325 g g-1 at the HRT of 12, 24, 48 h, respectively. Silveira et al. (2005) obtained a higher ethanol yield of 0.52 g g-1 with the

substrate fermentation and immobilized cells of *K. marxianus*.

85 - 148 g L-1 with fermentation time ranging from 29 to 64 h.

initial lactose concentration of 50 g L-1 with the yeast strain of *K. marxianus*.

Ethanol concentration ( g L-1)

Fig. 7. Effect of HRT on the ethanol yield.

Ozmihci & Kargi (2007) stated, that biomass concentration is an important parameter affecting the ethanol formation efficiency. The volumetric rate of sugar utilization can increase with biomass concentration. They found that when the biomass concentration was 510 mg L-1, the rate of sugar utilization was about 1580 mg L-1 h-1 at HRT of 30 h. The maximum sugar utilization rate of 2200 mg L-1 h-1 they obtained at the biomass concentration of 1020 mg L-1 and HRT of 24 h. In this study the concentration of yeast in the UASB reactors ranged from 705 to 869 mg L-1 by the duration of the experiment. The volumetric rate of sugar utilization was as high as 927, 1576, 1724 mg L-1 h-1 at HRT of 12, 24 and 48 h, respectively.
